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Altaicalarins A-D, Cytotoxic Bisabolane Sesquiterpenes from Ligularia altaica Qi Wang,† Tzu-Hsuan Chen,‡ Kenneth F. Bastow,‡ Kuo-Hsiung Lee,*,§ and Dao-Feng Chen*,† Department of Pharmacognosy, School of Pharmacy, Fudan UniVersity, Shanghai 201203, People’s Republic of China, DiVision of Medicinal Chemistry and Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, UniVersity of North Carolina, Chapel Hill, North Carolina 27599-7568 ReceiVed August 7, 2009
Four new bisabolane sesquiterpenes, altaicalarins A-D (1-4), and three known analogues (5-7) were isolated from the roots and rhizomes of Ligularia altaica. The structures were elucidated by spectroscopic methods including 2D NMR techniques, and the structure of 1 was confirmed by single-crystal X-ray diffraction. The isolated compounds were also evaluated for cytotoxic activity against human lung carcinoma (A-549), human breast adenocarcinoma (MCF7), epidermoid carcinoma of the nasopharynx (KB), and vincristine-resistant nasopharyngeal (KBVIN) cell lines, and 1 was found to show significant cytotoxicity, with EC50 values of 3.4, 0.8, 1.0, and 0.9 µg/mL, respectively. Ligularia altaica DC. (Asteraceae) is widely distributed in the Altaica mountains of China, and its roots and rhizomes are used as a folk medicine in the Xinjiang region, China, for easing breathing, stimulating blood flow, reducing inflammation, stopping cough, and eliminating phlegm.1 In prior investigations, flavonoids, triterpenes, sterols, benzofurans, and eremophilane sesquiterpenes were reported.2-4 As part of our program to discover anticancer agents from Chinese herbs, a phytochemical investigation of the roots and rhizomes of this plant led to the isolation and characterization of four new (1-4) and three known [1β,8-diangloyloxy-3β,4β,10,11diepoxybisabol-7(14)-ene (5),5 1β-acetoxy-1β,8-diangloyloxy10,11-dihydroxy-3,4-epoxybisabol-7(14)-ene (6),6 and bisabolone (7)7] bisabolane sesquiterpenes. Herein we report the isolation and structural elucidation of the new compounds, as well as their in vitro cytotoxicity evaluation of all isolates against human lung carcinoma (A-549), human breast adenocarcinoma (MCF-7), epidermoid carcinoma of the nasopharynx (KB), and vincristineresistant nasopharyngeal (KBVIN) cell lines.
Results and Discussion An EtOH extract of the roots and rhizomes of L. altaica was suspended in H2O and partitioned successively with petroleum ether, EtOAc, and n-BuOH. Repeated column chromatography of the combined EtOAc and petroleum ether portions on silica gel and RP18 gel, followed by preparative TLC, yielded four new (1-4) and three known (5-7) bisabolane sesquiterpenes. Compound 1 was obtained as a colorless solid, and its molecular formula was determined as C22H28O6 with nine degrees of unsaturation by HREIMS (m/z 388.1897). The IR spectrum showed absorption bands for OH (3423 cm-1), ester carbonyl (1740 cm-1), and double-bond (1694 cm-1) moieties. The 1H and 13C NMR spectra (Tables 1 and 2) displayed the characteristic signals of an angeloyloxy group [δH 6.08 (1H, qq, J ) 7.0, 1.5 Hz), 1.97 (3H, dq, J ) 7.0, 1.1 Hz), 1.90 (3H, dq, J ) 1.5, 1.1 Hz); δC 167.3 (C), 127.9 (C), 138.1 (CH), 15.7 (CH3), 20.6 (CH3)]8 and an acetoxy group [δH 2.30 (3H, s); δC 170.3 (C), 20.7 (CH3)], in accordance with the significant MS fragment peaks at m/z 288 [M - AngOH]+ and m/z 246 [M - AngOH - HOAc]+. Apart from these two ester groups, the 13C NMR and DEPT spectra of 1 revealed 15 skeletal * To whom correspondence should be addressed. Tel: +86-21-51980135. Fax: +86-21-51980017. E-mail:
[email protected] (D.F.C.) or khlee@ unc.edu (K.H.L.). † Fudan University. ‡ Division of Medicinal Chemistry, University of North Carolina at Chapel Hill. § Natural Products Research Laboratories, University of North Carolina at Chapel Hill.
10.1021/np900489h
carbon signals, including three methyl, two methylene, three methine, and seven quaternary carbons and the typical carbon signals for a terminal double bond [δC 144.4 (C) and 115.0 (CH2)]. The 1H NMR data of 1 displayed signals accounting for 18 protons: two hydroxy groups [δH 5.47, 5.58 (br s)], one aromatic [δH 2.11, 3H, s] and two allylic [δH 1.52, 1.65 (each 3H s)] methyls, two methylenes, including one exocyclic methylene [δH 5.09, 5.30 (each 1H, br s)], and three methines, including an oxymethine proton [δH 5.61, dd, J ) 7.8, 7.8 Hz], a trisubstituted olefinic proton [δH 5.07, dd, J ) 7.4, 1.5 Hz], and an aromatic proton [δH 6.36 (1H, s)]. HMBC correlations were found between the proton at δH 5.07 (1H, dd) and carbons at δC 25.7 (C-12) and 17.8 (C-13), the protons at δH 1.65 (3H, s) and 1.52 (3H, s) with the carbon at δC 119.2 (C-10), and the proton at δH 2.24 (1H, dd) with carbons at δC 119.2 (C-10) and 134.1 (C-11), which proved the presence of a prenyl group (Figure 1). The above spectroscopic data were in agreement with those reported for an aromatic bisabolane sesquiterpene with an angeloyloxy group, an acetoxy group, and two hydroxy groups.9 Furthermore, in the HMBC spectrum of 1 (Figure 1), the correlations of the proton at δH 5.61 (1H, d) with carbons at δC 167.3 (OAng, C-1′), 144.4 (C-7), 119.2 (C-10), 115.0 (C-14), and 32.2 (C-9) indicated attachment of the angeloyloxy group to C-8,
2010 American Chemical Society and American Society of Pharmacognosy Published on Web 01/14/2010
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Table 1. 1H NMR (400 MHz) Data of Compounds 1-4 (CDCl3, δ ppm, J in Hz) 1
2
1
6.48, d (7.8)
2 3 4 5 6 7 8 9a 9b 10 11 12 13 14a 14b 15 H-OH OAc OAng 3′ 3′′ 4′ 4′′ 5′ 5′′
6.63, d (7.8) 6.36, s
4 5.50, d (7.8)
5.70, d (12.9) 2.75, ddd (15.6, 12.9, 8.2)
5.70, d (11.7) 2.93, dd (11.7, 7.8)
3.45, s
5.61, dd (7.8, 7.8) 2.29, overlapped 2.24, dd (7.4, 7.4) 5.07, dd (7.4, 1.5)
5.09, dd (7.0, 7.0) 2.42, overlapped 2.40, overlapped 5.10, dd (7.4, 2.7)
5.10, dd (7.8, 6.2) 2.34, dd (6.6, 6.6) 2.34, dd (6.6, 6.6) 5.05, dd (7.1, 1.5)
5.43, dd (5.1, 5.1) 1.87, dd (5.9, 5.1) 1.83, dd (5.9, 4.7) 2.76, t (5.8)
1.65, s 1.52, s 5.30, br s 5.09, br s 2.11, s 5.47, 5.58, bl s 2.30, s
1.66, s 1.57, s 5.30, br s 5.15, br s 2.26, s 5.83, 9.00, br s
1.62, s 1.68, s 5.26, br s 5.12, br s 1.45, s
1.23, s 1.26, s 5.40, br s 5.31, br s 1.46, s
2.03, s
2.05, s
6.08, qq (7.0, 1.5)
6.25, qq (7.0, 1.5)
6.09, qq (7.0, 1.5)
1.97, dq (7.0, 1.1)
2.05, dq (7.0, 1.5)
1.98, dq (7.0, 1.1)
1.90, dq (1.5, 1.1)
1.96, dq (1.5, 1.5)
1.89, dq (1.5, 1.1)
6.11, qq (7.0, 1.5) 6.17, qq (7.4, 1.5) 1.98, dq (7.0, 1.1) 1.98, dq (7.4, 1.5) 1.90, dq (1.5, 1.1) 1.90, dq (1.5, 1.5)
Table 2. 13C NMR (100 MHz) Data of Compounds 1-4 (CDCl3, δ ppm) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OAc 1′ 2′ OAng 1′ 1′′ 2′ 2′′ 3′ 3′′ 4′ 4′′ 5′ 5′′
3 2.55, ddd (8.2, 7.4, 4.3) 2.20, ddd (15.6, 8.2, 7.4) 3.45, d (4.3)
1
2
3
4
129.7 (s) 152.1 (s) 112.3 (s) 146.5 (s) 107.9 (d) 130.7 (s) 144.4 (s) 74.8 (d) 32.2 (t) 119.2 (d) 134.1 (s) 25.7 (q) 17.8 (q) 115.0 (t) 8.5 (q)
120.2 (d) 121.6 (d) 124.0 (s) 142.9 (s) 140.5 (s) 123.2 (s) 147.3 (s) 76.5 (d) 33.2 (t) 119.1 (d) 134.7 (s) 25.7 (q) 17.9 (q) 114.8 (t) 15.4 (q)
31.8 (t) 64.5 (d) 61.4 (s) 201.5 (s) 74.8 (d) 43.8 (d) 147.5 (s) 75.6 (d) 32.0 (t) 118.9 (d) 134.5 (s) 25.7 (q) 18.0 (q) 113.0 (t) 14.8 (q)
71.6 (d) 65.6 (d) 61.4 (s) 199.5 (s) 72.8 (d) 48.5 (d) 145.5 (s) 72.6 (d) 33.5 (t) 60.8 (d) 58.3 (s) 24.6 (q) 18.8 (q) 114.8 (t) 14.3 (q)
169.7 (s) 20.2 (q)
169.4 (s) 20.2 (q) 166.0 (s) 166.2 (s) 126.6 (s) 127.3 (s) 139.6 (d) 140.9 (d) 15.8 (q) 15.9 (q) 20.5 (q) 20.4 (q)
170.3 (s) 20.7 (q) 167.3 (s)
169.8 (s)
166.8 (s)
127.9 (s)
127.1 (s)
127.8 (s)
138.1 (d)
140.9 (d)
138.6 (d)
15.6 (q)
15.9 (q)
15.7 (q)
20.6 (q)
20.3 (q)
20.5 (q)
whereas the correlations of the proton at δH 6.36 (1H, s) with carbons at δC 129.7 (C-1), 112.3 (C-3), and 146.5 (C-4) together with the correlations of the methyl protons at δH 2.11 (3H, s) with carbons at δC 152.1 (C-2), 112.3 (C-3), and 146.5 (C-4) indicated that the two hydroxy groups and an acetoxy group might be located at C-2, C-4, and C-1, respectively. However, the absolute configuration at C-8 could not be determined. The structure of 1 was further confirmed by X-ray crystallography (Figure 2). Therefore, 1 was established as 1-acetoxy-2,4-dihydroxy-8-angeloyloxybisabola-1,3,5,7(14),10(11)-pentaene, named altaicalarin A. The HREIMS of 2 gave m/z 330.1825 in accordance with the molecular formula C20H26O4 and eight unsaturation degrees. The IR spectrum showed absorptions for hydroxy (3260 cm-1), ester carbonyl (1703 cm-1), and double-bond (1674 cm-1) moieties. Most
of the NMR data of 2 were similar to those of 1 (Tables 1 and 2), except that signals for an acetoxy group were absent. 1H NMR signals for a pair of proton doublets [δH 6.63, 6.48 (each 1H, d, J ) 7.8 Hz)] and the HMBC correlations of the proton at δH 5.83 (H-OH) with carbons at δC 124.0 (C-3), 142.9 (C-4), and 140.5 (C-5) and of the proton at δH 9.00 (-OH) with carbons at C-4 and C-5 indicated that the two hydroxy groups should be located at C-4 and C-5, respectively (Figure 1). The configuration of 2 was the same as that of 1, because they both had a positive optical rotation. Thus, 2 was determined as 4,5-dihydroxy-8-angeloyloxybisabola-1,3,5,7(14),10(11)-pentaene, named altaicalarin B. Compound 3, colorless gum, has a molecular formula of C22H30O6 on the basis of HRESIMS data (m/z 413.1931 [M + Na]+). The 1H and 13C NMR spectra (Tables 1 and 2) displayed signals for angeloyloxy and acetoxy groups. The 13C NMR and DEPT spectra revealed 15 additional skeletal carbon signals: three methyl, three methylene, five methine, and four quaternary carbons. To accommodate 8 degrees of unsaturation, compound 3 was proposed to have a monocyclic sesquiterpene skeleton with a terminal double bond [δH 5.12, 5.26 (each 1H, br s); δC 113.0 (CH2) and 147.5 (C)], an epoxy group [δH 3.45 (1H, dd, J ) 4.3, 4.3 Hz); δC 64.5 (CH), 61.4 (C)], a carbonyl group (δC 201.3), an angeloyloxy group, and an acetoxy group. 1H-1H COSY 2D-NMR data indicated the two main structural sequences, -CH-CH2CH-CH- and -CH-CH2-CH-, which can be further connected with other fragments based on the following HMBC correlations: H-15/C-4; H-2/C-3; H-5/C-4, C-7; H-8/C-7; H-12, H-13/C-10 (Figure 3). Thus, 3 was determined to be a bisabolane sesquiterpene.10 In the HMBC spectrum of 3, correlations of the proton at δH 5.70 (1H, d) with carbons at δC 169.7 (OAc), 201.3 (C-4), and 43.8 (C-6) and of the proton at δH 5.10 (1H, dd) with carbons at δC 166.8 (OAng, C-1′), 147.5 (C-7), 118.9 (C-10), 113.0 (C-14), and 32.0 (C-9) indicated that the acetoxy and angeloyloxy groups are attached to C-5 and C-8, respectively. The HMBC correlations of the proton at δH 3.45 (1H, d) with carbons at δC 31.8 (C-1), 61.4 (C-3), and 43.8 (C-6) indicated that the epoxy group should be assigned at C-2 and C-3. The location of the carbonyl group at C-4 was established by HMBC correlations of the protons at δH 1.45 (3H, s) and 5.70 (1H, d) with the carbon at δC 201.3 (C-4). The stereostructure of 3 was deduced from the 1H NMR coupling constants and the ROESY spectrum (Figure 3). When H-6 is
Cytotoxic Bisabolane Sesquiterpenes from Ligularia
Figure 1. Key HMBC correlations of 1 and 2. assumed to be in an R-orientation,11 H-5 should be β-axial due to a large 3J5, 6 value (12.9 Hz), and the proton at δH 2.25 should be H-1 and R-oriented due to a smaller 3J1, 6 value (8.2 Hz). In the ROESY spectrum, the obvious correlation of H-2 with H-1R and H-15 revealed that the epoxy group should be 2β,3β-oriented. The relative configuration at C-8 could not be determined from the spectroscopic data. Thus, the structure of 3 was determined as 5Racetoxy-8-angeloyloxy-2β,3β-epoxybisabola-7(14),10(11)-dien-4one, named altaicalarin C. The molecular formula of 4 was determined as C27H36O9 on the basis of the HRESIMS (m/z 527.2250 [M + Na]+). The IR spectrum indicated the presence of ketone (1755 cm-1) and ester carbonyl groups (1720 cm-1) and a double bond (1647 cm-1). The 1H and 13 C NMR spectra (Tables 1 and 2) displayed signals for one acetoxy and two angeloyloxy groups. Apart from the three ester groups, the 1H and 13C NMR and 1H-1H COSY spectra (Figure 3) of 4 indicated that the basic skeleton of compound 4 was similar to that of 3, except for an epoxy group [δH 2.76 (1H, t, J ) 5.8 Hz); δC 60.8 (CH), 58.3 (C)] at C-10 and C-11 in 4, rather than a corresponding trisubstituted double bond in 3. The locations of the two epoxy groups at C-2, C-3 and C-10, C-11 were confirmed by HMBC correlations of the proton at δH 3.45 (1H, s) with carbons at δC 71.6 (C-1), 61.4 (C-3), and 48.5 (C-6) and of the proton at δH 2.76 (1H, dd) with carbons at δC 72.6 (C-8), 33.5 (C-9), and 24.6 (C-12). The HMBC correlations of the proton at δH 5.70 (1H, d) with carbons at δC 169.4 (OAc), 199.5 (C-4), and 48.5 (C-6), the proton at δH 5.50 (1H, d) with carbons at δC 166.2 (OAng), 65.6 (C-2), 61.4 (C-3), and 72.8 (C5), and the proton at δH 5.43 (1H, dd) with carbons at δC 166.0 (OAng), 145.5 (C-7), and 60.8 (C-10) indicated that the acetoxy group is at C-5 and the two angeloyloxy groups are at C-1 and C-8 (Figure 3).
Figure 2. X-ray crystal structure of 1.
Journal of Natural Products, 2010, Vol. 73, No. 2 141
Figure 3. Key HMBC, 1H-1H COSY, and ROESY correlations of 3 and 4. Table 3. Cytotoxicity Data of Compounds 1-7 (EC50 µg/mL) cell line compound
A549
MCF-7
KB
KBVIN
1 2 3 4 5 6 7 etoposide
3.4 >20 11.4 >20 >20 >20 >20 0.4
0.8 10.8 7.7 >20 >20 >20 >20 16.4
1.0 11.8 10.4 >20 >20 >20 >20 3.9
0.9 15.9 7.6 18.9 11.4 >20 >20 8.8
The relative configuration of the stereogenic centers of the cyclohexane ring in 4 was the same as those in 3, on the basis of the 1H NMR coupling constants (J5,6 ) 11.7, J6,1 ) 7.8 Hz) and the ROESY correlations between H-1/H-2 and H-2/H-15. The relative configurations at C-8 and C-10 could not, however, be determined from the spectroscopic data. Thus, the structure of 4 was determined as 1β,8-diangeloyloxy-5R-acetoxy-2β,3β;10,11diepoxybisabola-7(14)-en-4-one, named altaicalarin D. All isolates were evaluated for their in vitro cytotoxicity against human lung carcinoma (A-549), human breast adenocarcinoma (MCF-7), epidermoid carcinoma of the nasopharynx (KB), and vincristine-resistant nasopharyngeal (KBVIN) cell lines according to a described procedure.12 The results are listed in Table 3. The most potent compound, 1, exhibited significant cytotoxicity against all four cell lines with EC50 values of 3.4 (A549), 0.8 (MCF-7), 1.0 (KB), and 0.9 (KBVIN) µg/mL. Compound 2, which differs from 1 in the number and position of the aromatic hydroxy and ester groups, showed only weak cytotoxicity against the latter three
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Journal of Natural Products, 2010, Vol. 73, No. 2
cancer cell lines. Among the compounds without an aromatic ring, 6 and 7 showed no activity (EC50 >20 µg/mL) against the cell line panel, 3 showed weak activity against all four cell lines, while 4 and 5 showed weak activity only against KBVIN. Generally, the compounds with a C-10-C-11 double bond (1-3) showed broader cytotoxicity compared with compounds with a C-10-C-11 epoxy group (4, 5). Experimental Section General Experimental Procedures. Melting points were measured on an XT-4 micromelting point apparatus and are uncorrected. Optical rotations were recorded on a JASCO P-1020 polarimeter at room temperature. UV spectra were measured on a Shimadzu UV-260 spectrophotometer in absolute MeOH. IR spectra were recorded on an Avatar 360 FT-IR ESP spectrometer in CH2Cl2. Mass spectra were determined on an HP5989A mass spectrometer for EIMS, a Waters Micromass GCT mass spectrometer for HREIMS, and a Bruker Daltonics APEXIII 7.0 TESLA FTMS mass spectrometer for HRESIMS. 1H and 13C NMR spectra were recorded on a Bruker DRX400 spectrometer in CDCl3. Analytical and preparative TLC were run on silica gel plates (GF254, Yantai Institute of Chemical Technology, Yantai, China). Spots were observed under UV light and visualized by spraying with 10% H2SO4, followed by heating. Column chromatography was performed on silica gel (200-300 and 300-400 mesh; Qingdao Marine Chemical Factory, Qingdao, China) and Lichroprep RP18 gel (40-60 µm, Merck, Darmstadt, Germany). X-ray crystallographic analysis was carried out on a Bruker Smart Apex CCD diffractometer with graphite-monochromated Mo KR radiation (λ ) 0.71073 Å). The structure was solved by direct methods using the program SHELXS. Non-hydrogen atoms were refined anisotropically. Hydrogen atoms were located by geometry and riding on the related atoms during refinements with a temperature factor of 1.2 or 1.5 times the latter’s. Plant Material. The roots and rhizomes of L. altaica were collected in August 2005 on the Altaica mountains (altitude 2000 m) in Xinjiang, China. The identity of the plant material was verified by Professor Ping Yan at Shihezi University, and a voucher specimen (WQ-LA-05-2) has been deposited in the Herbarium of Materia Medica, Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai, People’s Republic of China. Extraction and Isolation. The dried and powdered materials (5.0 kg) were extracted three times with 95% EtOH at reflux temperature and filtered. The filtrate was evaporated in vacuo to give a residue (360 g), a portion of which (350 g) was suspended in H2O (2 L) and partitioned successively with petroleum ether (3 × 1.5 L) and EtOAc (3 × 1.5 L). The combined EtOAc (160 g) and petroleum ether (50 g) extracts were chromatographed on a Si gel (200-300 mesh, 2 kg, 10 × 120 cm) column, eluted successively with petroleum ether-acetone (50:1, 30:1, 15:1, 9:1, 7:1, 5:1, 3:1, 2:1, 1:1) to yield fractions 1-6. Subsequently, fractions 1, 2, 3, 5, and 6 were subjected to silica gel CC with petroleum ether-EtOAc (80:1, 25:1, 15:1, 5:1, 3:1), respectively. Fraction 1 (26 g) yielded 7 (740 mg). Fraction 2 (28 g) gave two fractions, 2a and 2b. Fraction 2a (16 g) was applied to silica gel CC with petroleum ether-EtOAc (30:1) to afford 5 (287 mg), and fraction 2b (6 g) was chromatographed similarly, followed by CC on RP18 gel with MeOH-H2O (80:20) to give 2 (25 mg). Fraction 3 (27 g) gave two fractions, 3a and 3b. Fraction 3b (7 g) was applied to silica gel CC with petroleum ether-EtOAc (9:1) to afford 4 (68 mg). Fraction 5 (25 g) gave two fractions, 5a and 5b. Fraction 5a (5 g) was applied to silica gel CC with petroleum ether-EtOAc (6:1) to give 6 (262 mg), and fraction 5b (6 g) was applied to silica gel CC with petroleum ether-EtOAc (5:1), followed by preparative TLC with petroleum ether-acetone (12:1) to give 3 (138 mg). Fraction 6 (39 g) afforded 1 (66 mg), after further purification over preparative TLC with CHCl3-acetone (20:1). 1-Acetoxy-2,4-dihydroxy-8-angeloyloxybisabola-1,3,5,7(14),10(11)pentaene (1): colorless blocks (acetone); mp 122-123 °C; [R]22 D +129 (c 0.3, MeOH); IR νmax (KBr) 3423, 1740, 1694, 1644, 1434, 1239, 1098 cm-1; UV λmax (MeOH) nm (log ε) 284 (sh), 211 (1.36); 1H NMR (CDCl3) and 13C NMR (CDCl3) spectroscopic data, see Tables 1 and 2; EIMS m/z 388 [M]+ (2.6), 288 [M - AngOH]+ (26.7), 246 [M - AngOH - HOAc]+ (97.0), 231 (60.6), 177 (100), 83 (39.1), 55 (30.3); HREIMS m/z 388.1897 (calcd for C22H28O6, 388.1886). Crystal data:13 C22H28O6, Mr ) 388.44, monoclinic, space group C2, a ) 17.066(4) Å, b )
Wang et al. 6.9212(15) Å, β ) 100.536(4)°, c ) 19.004(4) Å, V ) 2206.9(8) Å3, Z ) 4, Dcalc ) 1.169 Mg/m3. The final R values were R1 ) 0.0564 and wR2 ) 0.1220 for 6925 observed reflections [I > 2σ(I)]. 4,5-Dihydroxy-8-angeloyloxybisabola-1,3,5,7(14),10(11)-pentaene (2): colorless gum, [R]22 D +23.3 (c 0.6, MeOH); IR νmax (CH2Cl2) 3260, 2921, 1703, 1674, 1463, 1257, 739 cm-1; UV λmax (MeOH) nm (log ε) 280 (sh), 210 (1.26); 1H NMR (CDCl3) and 13C NMR (CDCl3) spectroscopic data, see Tables 1 and 2; EIMS m/z 330 [M]+ (2.3), 230 [M - AngOH]+ (33.7), 215 [M - AngOH - Me]+ (42.3), 193 [M AngOH - H - 2 × H2O]+ (9.4), 161 (100), 83 (68.8), 55 (87.1); HREIMS m/z 330.1825 (calcd for C20H26O4, 330.1831). 5r-Acetoxy-8-angeloyloxy-2β,3β-epoxybisabola-7(14),10(11)-dien4-one (3): colorless gum, [R]22 D -26.3 (c 0.5, MeOH); IR νmax (CH2Cl2) 1735, 1646, 1459, 1378, 1142, 736 cm-1; 1H NMR (CDCl3) and 13C NMR (CDCl3) spectroscopic data, see Tables 1 and 2; EIMS m/z 331 (2.7), 228 (2.9), 173 (10.1), 97 (100); HRESIMS m/z 413.1931 [M + Na]+ (calcd for C22H30O6Na, 413.1934). 1β,8-Diangeloyloxy-5r-acetoxy-2β,3β;10,11-diepoxybisabola-7(14)en-4-one (4): colorless gum, [R]D22 +44.4 (c 0.2, MeOH); IR νmax (CH2Cl2) 1757, 1720, 1647, 1457, 1378, 1230, 1148, 1040, 754 cm-1; 1 H NMR (CDCl3) and 13C NMR (CDCl3) spectroscopic data, see Tables 1 and 2; EIMS m/z 405 [M + H - AngOH]+ (2.7), 263 [M + H 2AngOH - HOAc]+ (1.2), 219 (4.6), 83 (100); HRESIMS m/z 527.2250 [M + Na]+ (calcd for C27H36O9Na, 527.2251). Growth Inhibition Assay. Drug stock solutions were prepared in DMSO and stored at -70 °C. Upon dilution into culture medium, the final DMSO concentration was e1% DMSO (v/v), a concentration without effect on cell replication. The human tumor cell line panel consisted of lung carcinoma (A-549), breast adenocarcinoma (MCF7), epidermoid carcinoma of the nasopharynx (KB), and vincristineresistant nasopharyngeal (KBVIN). Etoposide was used as control. Cell culture and other procedures were the same as those reported previously.12 The EC50 value is the concentration that inhibited growth by 50% following two days of continuous exposure. Acknowledgment. This investigation was supported by grants from the State Key Program (2009ZX09502-013, 2009ZX09301-011) from the Ministry of Science and Technology, the National Natural Science Foundation for Excellent Youth (30925042), People’s Republic of China (to D.F.C.), Fudan University Graduate Innovation Foundation (CQF301812) (to Q.W.), and Grant CA-17625 from the National Cancer Institute, NIH (to K.H.L.). Supporting Information Available: NMR spectra of the new compounds 1-4, as well as crystallographic data of 1. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) National Institute for the Control of Pharmaceutical and Biological Products. Chinese Folk Materia Medica; People Health Publishing House: Beijing, 1990; Vol. 2, pp 571-590. (2) Volkhonskaya, T. A.; Khanminchun, V. M.; Frolova, O. I. Rastit. Resur. 1983, 19, 455–464. (3) Zhang, C. F.; Wang, Q.; Zhang, M. Chin. Pharm. J. 2008, 43, 1697– 1700. (4) Zhang, C. F.; Wang, Q.; Zhang, M. Chin. Med. Mater. 2009, 32, 524– 526. (5) Liu, C. M.; Fei, D. Q.; Wu, Q. H.; Gao, K. J. Nat. Prod. 2006, 69, 695–699. (6) Liao, J. C.; Yang, L.; Jia, Z. J. Chem. Res. Chin. UniV. 1999, 15, 67–69. (7) Andersen, N. H.; Syrdal, D. D. Phytochemistry 1970, 9, 1325–1340. (8) Bohlmann, F.; Zdero, C.; Nagabushan, R. Chem. Ber. 1972, 105, 3523– 3531. (9) Bohlmann, F.; Zdero, C. Phytochemistry 1978, 17, 1591–1593. (10) Zhu, Y.; Yang, L.; Jia, Z. J. J. Nat. Prod. 1999, 62, 1479–1483. (11) Onuki, H.; Yamazaki, M.; Nakamura, A.; Hana, i R.; Kuroda, C.; Gong, X.; Shen, Y. M.; Hirota, H. J. Nat. Prod. 2008, 71, 520–524. (12) Cheng, H. H.; Wang, H. K.; Ito, J.; Bastow, K. F.; Tachibana, Y.; Nakanishi, Y.; Xu, Z.; Luo, T. Y.; Lee, K. H. J. Nat. Prod. 2001, 64, 915–919. (13) Crystallographic data for the structure reported in this paper have been deposited with the Cambridge Crystallographic Data Center. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44(0)1223-336033 or e-mail:
[email protected]).
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